Composition containing a bean extract for improving blood circulation and  increasing vascular health

ABSTRACT

The present invention relates to a composition containing a bean extract extracted by low-concentration, low-grade alcohol or fractions thereof. The composition exhibits excellent effects in improving blood circulation, improving obesity, and preventing diabetes, hyperlipidemia and the like, and exhibits the effects of alleviating or treating the symptoms of diabetes, hyperlipidemia, and the like. The present invention also relates to a method for improving blood circulation and vascular health.

TECHNICAL FIELD

The present disclosure relates to a composition containing a beanextract for improving blood circulation and vascular health.

BACKGROUND ART

Modern people take in more fats with the change in eating habits, butthey tend to exercise less and suffer from various stresses. With thechange in the dietary lives, various diseases including hypertension,arteriosclerosis and blood circulation disorder are increasing. Inparticular, the blood circulation disorder is known to cause suchsymptoms as decline of memory, lethargy, lack of concentration, chronicfatigue, and the like.

Blood circulation refers to the flow of blood in the body in specificdirections, and the blood circulation disorder refers to a condition inwhich the blood vessels become inelastic and cholesterol, etc. isdeposited in the inner wall of the blood vessels, leading to narrowingof the blood vessels and interrupting blood circulation.

The diseases caused by the blood circulation disorder includecardiovascular diseases such as hyperlipidemia, arteriosclerosis,myocardial infarction, cerebral thrombosis, etc. Among thecardiovascular diseases, hypertension, arteriosclerosis, heart diseaseand stroke are one of the most important causes of death in the elderlypeople.

As such, if the blood circulation disorder is left untreated, it willlead to difficulties in maintaining normal lives and, in severe cases,various types of diseases and even death. Accordingly, it is thoughtthat prevention is more important than treatment of the diseases causedby the blood circulation disorder. Although drugs for cardiovasculardiseases are used clinically at present, they are expensive and maycause various side effects.

DISCLOSURE Technical Problem

The present disclosure is directed to providing a composition forimproving blood circulation.

The present disclosure is also directed to providing a composition forimproving vascular health.

The present disclosure is also directed to providing a pharmaceuticalcomposition for improving blood circulation.

The present disclosure is also directed to providing a pharmaceuticalcomposition for improving vascular health.

The present disclosure is also directed to providing a health foodcomposition for improving blood circulation.

The present disclosure is also directed to providing a health foodcomposition for improving vascular health.

Technical Solution

In one general aspect, the present disclosure provides a compositioncontaining a bean extract extracted with a low-concentration, loweralcohol or a fraction thereof as an active ingredient.

Advantageous Effects

The composition according to the present disclosure has superior effectof improving blood circulation and is effective for prevention,amelioration or treatment of cardiovascular diseases, including obesity,diabetes, hyperlipidemia, etc.

Best Mode

The previous studies on beans are concentrated on isolation andpurification of pharmacologically active ingredients from bean, andstudies about the medical use of the bean itself are insufficient. Inaddition, the commonly employed extraction method was one using ahigh-concentration organic solvent.

A bean extract contains a lot of ingredients not known as yet, and someof them exhibit useful pharmacological effect for the human body. Theinventors of the present disclosure have acquired a bean extract using alow-concentration, lower alcohol as extraction solvent, unlike in theextraction method commonly employed for extraction of natural productsor herbs, and have shown that the extract exhibits strongerantithrombotic effect than one extracted using a high-concentrationorganic solvent.

A composition according to an embodiment of the present disclosurecomprises a bean extract extracted using a low-concentration organicsolvent or a fraction of the extracted bean extract. In an exemplaryembodiment, the organic solvent may be a C₁-C₅ alcohol, although notbeing limited thereto. The C₁-C₅ alcohol may be, for example, at leastone selected from a group consisting of methanol, ethanol, isopropylalcohol, n-propyl alcohol, n-butanol and isobutanol, specificallyethanol. In another exemplary embodiment, the concentration of the C₁-C₅alcohol may be 1-70% (v/v), specifically 1-40% (v/v), more specifically5-25% (v/v), more specifically 7-20% (v/v). For example, the solvent maybe 10% or 20% (v/v) ethanol.

The composition according to the present disclosure is obtained byextracting bean using the low-concentration, lower alcohol. Throughvarious researches and repeated experiments, the inventors of thepresent disclosure have identified that a bean extract extracted using alower alcohol among various organic solvents, for example, ethanol,particularly low-concentration ethanol, has the effect of improvingblood circulation and vascular health and thus being useful inalleviation or treatment of obesity, diabetes or hyperlipidemia.

The fraction of the bean extract refers to a component isolated fromfurther fractionation of the bean extract. In an exemplary embodiment,the fraction of the bean extract may be an ethyl acetate or butanolfraction, specifically an ethyl acetate fraction, of the C₁-C₅ alcoholextract. The inventors of the present disclosure have obtained variousfractions from the bean extract extracted using the low-concentrationethanol. As a result, an ethyl acetate or butanol fraction showed bettereffect of improving blood circulation than a water fraction. Especially,the ethyl acetate fraction showed a very superior effect.

The composition comprising the bean extract or the fraction thereofaccording to the present disclosure has an effect of inhibiting bloodclotting by suppressing platelet aggregation as well as an effect ofinducing vasodilation by suppressing constriction of blood vessels.Further, the composition has an effect of reducing lipids in blood andthe liver by suppressing increase of cholesterol. Accordingly, thecomposition is effective in improving blood circulation and vascularhealth and may be effectively used to treat or prevent obesity,diabetes, hyperlipidemia, or the like.

In an exemplary embodiment, the bean extract or the fraction thereof maycomprise adenosine as a marker or functional component. The inventors ofthe present disclosure have separated and purified the bean extract orthe fraction thereof. After measuring activities of the separated andpurified products, it was confirmed that adenosine is included therein.In an exemplary embodiment, adenosine may be included in an amount of0.01-1.0 wt %, more specifically 0.1-0.6 wt %, based on the weight ofthe bean extract or the fraction thereof. That is to say, the beanextract extracted using the low-concentration, lower alcohol accordingto the present disclosure contains relatively large amount of activeingredients such as adenosine.

The bean is not particularly limited as long as the bean extract or thefraction thereof has an effect of improving blood circulation. In anexemplary embodiment, the bean may be black bean or colored bean. Inanother exemplary embodiment, the bean may be at least one selected froma group consisting of Seoritae (Glycin max MERR), Seomoktae (RhynchosiaNolubilis), Black soybean (Glycine max(L.) Merr.), blue bean (Glycimemax MERR), yellow bean (Glycime max MERR), field bean (Vicia faba),kidney bean (Phaseolus vulgaris), pinto bean (Phaseolus vulgaris L.),small red bean (Vigna angularis), small black bean (Phaseolus angularis.F.WIGHT.), sprouting bean (Glycine max (L.) Merr.) and soybean (Glycinemax). More specifically, the bean may be seoritae or field bean.

As used herein the term “black bean” collectively refers to a bean whosegrain exhibits black color. The black bean is not particularly limited.For example, it may be black bean (Glycine max), Seoritae (Glycin maxMERR), Seomoktae (Rhynchosia Nolubilis), Black soybean (Glycine max(L.)Merr.), or the like. The black bean may be called differently indifferent regions. And, the term “black bean extract” collectivelyrefers to a substance extracted from black bean. For example, itincludes a substance extracted using an organic solvent and also coversvarious fractions of the extract.

Also, as used herein the term “colored bean” collectively refers to abean whose grain exhibits deep color, not only black color but also red,yellow or blue color. Examples of the colored bean include Seoritae(Glycin max MERR), Seomoktae (Rhynchosia Nolubilis), Black soybean(Glycine max(L.) Merr.), blue bean (Glycime max MERR), yellow bean(Glycime max MERR), field bean (Vicia faba), kidney bean (Phaseolusvulgaris), pinto bean (Phaseolus vulgaris L.), small red bean (Vignaangularis), small black bean (Phaseolus angularis .F.WIGHT), sproutingbean (Glycine max (L) Merr.) and soybean (Glycine max), although notbeing limited thereto. The colored bean may be called differently indifferent regions. And, the term “colored bean extract” collectivelyrefers to a substance extracted from colored bean. For example, itincludes a substance extracted using an organic solvent and also coversvarious fractions of the extract.

The present disclosure provides a pharmaceutical composition comprisingthe afore-described composition. In an exemplary embodiment, thepharmaceutical composition may be a pharmaceutical composition forimproving blood circulation, preventing cardiovascular disease, oralleviating or treating related symptoms. The pharmaceutical compositionaccording to the present disclosure has the effect of preventing bloodclotting, suppressing vasoconstriction and/or reducing cholesterol.Specifically, the pharmaceutical composition may be a pharmaceuticalcomposition for improving blood circulation via antithrombotic effect,or alleviating or treating cardiovascular diseases including obesity,diabetes, hyperlipidemia, etc. The cardiovascular diseases may include,for example, obesity, diabetes, stroke, cerebral hemorrhage,arteriosclerosis, angina, myocardial infarction, hypertension, anemia,migraine, hyperlipidemia, or the like.

When the composition according to the present disclosure is used as amedicine, it may be prepared in the form of solid, semisolid or liquidby adding a commonly used organic or inorganic carrier for oral orparenteral administration.

A formulation for oral administration may be in the form of tablet,pill, granule, soft/hard capsule, powder, fine granule, dust, emulsion,syrup, pellet, or the like. A formulation for parenteral administrationmay be in the form of injection, drip, ointment, lotion, spray,suspension, emulsion, suppository, or the like. The active ingredient ofthe present disclosure may be easily prepared into such formulationaccording to a commonly employed method, and commonly used adjuvantssuch as surfactant, excipient, colorant, fragrance, preservative,stabilizer, buffering agent, suspending agent, etc. may be adequatelyused.

The pharmaceutical composition may be administered orally orparenterally, e.g., rectally, topically, transdermally, intravenously,intramuscularly, intraabdominally or subcutaneously.

An administration dose of the active ingredient will vary depending onthe age, sex and body weight of the subject, particular disease orpathological condition to be treated, severity of the disease orpathological condition, administration route, or decision by aphysician. The determination of the administration dose based on suchfactors is within the knowledge of those skilled in the art. A generaladministration dose is 0.001-2000 mg/kg/day, more specifically 0.5-1500mg/kg/day.

In an exemplary embodiment, the present disclosure provides a foodadditive, a functional food or a health food comprising the compositionaccording to the present disclosure. Specifically, the composition maybe a health food composition for improving blood circulation, oralleviating or treating cardiovascular diseases including obesity,diabetes, hyperlipidemia, etc. The cardiovascular diseases may include,for example, obesity, diabetes, stroke, cerebral hemorrhage,arteriosclerosis, angina, myocardial infarction, hypertension, anemia,migraine, hyperlipidemia, or the like.

The present disclosure provides various types of food additive orfunctional food comprising the afore-described composition according tothe present disclosure. The composition may be processed into fermentedmilk, cheese, yogurt, juice, probiotic, dietary supplement or other foodadditives.

In an exemplary embodiment, the composition may further comprise otheringredients providing synergic effect within the range not negativelyaffecting the main effect desired by the present disclosure. Forexample, it may further comprise such additives as fragrance, pigment,sterilizer, antioxidant, antiseptic, humectant, thickener, mineral,emulsifier, synthetic polymer, etc. for improvement of physicalproperties. In addition, it may further comprise such auxiliaryingredients as water-soluble vitamin, oil-soluble vitamin, polypeptide,polysaccharide, seaweed extract, or the like. Those skilled in the artwill select and mix these ingredients without difficulty considering theformulation type or purpose of use, and their content may be determinedwithin the range not negatively affecting the purpose and effect of thepresent disclosure. For example, those ingredients may be added in anamount of 0.01-5 wt %, more specifically 0.01-3 wt %, based on the totalweight of the composition.

The composition according to the present disclosure may be in variousforms, including solution, emulsion, viscous mixture, tablet, powder,etc., and may be administered in various manners, including drinking,injection, spraying, squeezing, etc.

MODE FOR INVENTION

The examples and experiments will now be described. The followingexamples and experiments are for illustrative purposes only and notintended to limit the scope of this disclosure.

EXAMPLE 1 Preparation of 10% Ethanol Seoritae (Glycin max MERR) Extract

Dried seoritae (1 kg) was immersed in 10% ethanol solution (10 L) at 50°C. After extracting 3 times for 5 hours under reflux and allowing tostand at room temperature for 12 hours, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 7-20% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 2 Preparation of 20% Ethanol Seoritae (Glycin max MERR) Extract

Dried seoritae (1 kg) was immersed in 20% ethanol solution (10 L) at 50°C. After extracting 3 times for 5 hours under reflux and allowing tostand at room temperature for 12 hours, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 7-20% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 3 Preparation of 50% Ethanol Seoritae (Glycin max MERR) Extract

Dried seoritae (1 kg) was immersed in 50% ethanol solution (10 L) at 50°C. After extracting 3 times for 5 hours under reflux and allowing tostand at room temperature for 12 hours, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 7-20% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 4 Preparation of 70% Ethanol Seoritae (Glycin max MERR) Extract

Dried seoritae (1 kg) was immersed in 70% ethanol solution (10 L) at 50°C. After extracting 3 times for 5 hours under reflux and allowing tostand at room temperature for 12 hours, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 7-20% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 5 Preparation of 20% Ethanol Seomoktae (Rhynchosia Nolubilis)Extract

Dried seomoktae (1 kg) was immersed in 20% ethanol solution (5 L) at 60°C. After extracting for 3 hours under reflux and allowing to stand atroom temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 6 Preparation of 20% Ethanol Kidney Bean (Phaseolus vulgaris)extract

Dried kidney bean (300 g) was immersed in 20% ethanol solution (1.5 L)at 60° C. After extracting for 3 hours under reflux and allowing tostand at room temperature for a predetermined time, the extract wasfiltered, concentrated under reduced pressure and lyophilized to preparea powder sample. The yield was 3-15% and the prepared powder was kept atlow temperature until use.

EXAMPLE 7 Preparation of 20% Ethanol Pinto Bean (Phaseolus vulgaris L.)Extract

Dried pinto bean (300 g) was immersed in 20% ethanol solution (1.5 L) at60° C. After extracting for 3 hours under reflux and allowing to standat room temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 8 Preparation of 20% Ethanol Sprouting Bean (Glycine max (L.)Merr.) Extract

Dried sprouting bean (300 g) was immersed in 20% ethanol solution (1.5L) at 60° C. After extracting for 3 hours under reflux and allowing tostand at room temperature for a predetermined time, the extract wasfiltered, concentrated under reduced pressure and lyophilized to preparea powder sample. The yield was 3-15% and the prepared powder was kept atlow temperature until use.

EXAMPLE 9 Preparation of 20% Ethanol Yellow Bean (Glycime max MERR)Extract

Dried yellow bean (1 kg) was immersed in 20% ethanol solution (5 L) at60° C. After extracting for 2 times for 3 hours under reflux andallowing to stand at room temperature for a predetermined time, theextract was filtered, concentrated under reduced pressure andlyophilized to prepare a powder sample. The yield was 3-15% and theprepared powder was kept in a refrigerator until use.

EXAMPLE 10 Preparation of 20% Ethanol Blue Bean (Glycime max MERR)Extract

Dried blue bean (300 g) was immersed in 20% ethanol solution (1.5 L) at60° C. After extracting for 3 hours under reflux and allowing to standat room temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept in arefrigerator until use.

EXAMPLE 11 Preparation of 20% Ethanol Field Bean (Vicia faba) Extract

Dried field bean (1 kg) was immersed in 20% ethanol solution (5 L) at60° C. After extracting for 3 hours under reflux and allowing to standat room temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 12 Preparation of 20% Ethanol Soybean (Glycine max) Extract

Dried soybean (1 kg) was immersed in 20% ethanol solution (5 L) at 60°C. After extracting for 3 hours under reflux and allowing to stand atroom temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 13 Preparation of 20% Ethanol Small Black Bean (Phaseolusangularis .F.WIGHT.) Extract

Dried geodu (300 g) was immersed in 20% ethanol solution (1.5 L) at 60°C. After extracting for 3 hours under reflux and allowing to stand atroom temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept in arefrigerator until use.

EXAMPLE 14 Preparation of 20% Ethanol Small Red Bean (Vigna angularis)Extract

Dried red bean (300 g) was immersed in 20% ethanol solution (1.5 L) at60° C. After extracting for 3 hours under reflux and allowing to standat room temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 15 Preparation of 20% Ethanol Black Soybean (Glycine max(L.)Merr.) Extract

Dried heuktae (300 g) was immersed in 20% ethanol solution (1.5 L) at60° C. After extracting for 3 hours under reflux and allowing to standat room temperature for a predetermined time, the extract was filtered,concentrated under reduced pressure and lyophilized to prepare a powdersample. The yield was 3-15% and the prepared powder was kept at lowtemperature until use.

EXAMPLE 16 Preparation of Seoritae (Glycin max MERR) Butanol Extract

The 20% ethanol seoritae extract (25 g) obtained in Example 2 wasdissolved in distilled water (250 mL). After extracting 2 times withn-butanol (250 mL) using a separatory funnel, the resulting butanollayer was concentrated under reduced pressure and lyophilized to preparea sample. The yield was 5-15% and the prepared sample was kept in arefrigerator until use.

EXAMPLE 17 Preparation of Seomoktae (Rhynchosia Nolubilis) ButanolExtract

The 20% ethanol seomoktae extract (1 g) obtained in Example 5 wasdissolved in distilled water (10 mL). After extracting 2 times withn-butanol (10 mL) using a separatory funnel, the resulting butanol layerwas concentrated under reduced pressure and lyophilized to prepare asample. The yield was 5-15% and the prepared sample was kept in arefrigerator until use.

EXAMPLE 18 Preparation of Field Bean (Vicia faba) Butanol Extract

The 20% ethanol field bean extract (25 g) obtained in Example 11 wasdissolved in distilled water (250 mL). After extracting 2 times withn-butanol (250 mL) using a separatory funnel, the resulting butanollayer was concentrated under reduced pressure and lyophilized to preparea sample. The yield was 5-15% and the prepared sample was kept in arefrigerator until use.

EXAMPLE 19 Preparation of Blue Bean (Glycime max MERR) Butanol Extract

The 20% ethanol blue bean extract (1 g) obtained in Example 10 wasdissolved in distilled water (10 mL). After extracting 2 times withn-butanol (10 mL) using a separatory funnel, the resulting butanol layerwas concentrated under reduced pressure and lyophilized to prepare asample. The yield was 5-15% and the prepared sample was kept at lowtemperature until use.

EXAMPLE 20 Preparation of Yellow Bean (Glycime max MERR) Butanol Extract

The 20% ethanol yellow bean extract (1 g) obtained in Example 9 wasdissolved in distilled water (10 mL). After extracting 2 times withn-butanol (10 mL) using a separatory funnel, the resulting butanol layerwas concentrated under reduced pressure and lyophilized to prepare asample. The yield was 5-15% and the prepared sample was kept at lowtemperature until use.

EXAMPLE 21 Preparation of Sprouting Bean (Glycine max (L.) Merr.)Butanol Extract

The 20% ethanol sprouting bean extract (1 g) obtained in Example 8 wasdissolved in distilled water (10 mL). After extracting 2 times withn-butanol (10 mL) using a separatory funnel, the resulting butanol layerwas concentrated under reduced pressure and lyophilized to prepare asample. The yield was 5-15% and the prepared sample was kept at lowtemperature until use.

EXAMPLE 22 Preparation of Soybean (Glycine max) Butanol Extract

The 20% ethanol soybean extract (25 g) obtained in Example 12 wasdissolved in distilled water (250 mL). After extracting 2 times withn-butanol (250 mL) using a separatory funnel, the resulting butanollayer was concentrated under reduced pressure and lyophilized to preparea sample. The yield was 5-15% and the prepared sample was kept at lowtemperature until use.

EXAMPLE 23 Preparation of Kidney Bean (Phaseolus vulgaris) ButanolExtract

The 20% ethanol kidney bean extract (1 g) obtained in Example 6 wasdissolved in distilled water (10 mL). After extracting 2 times withn-butanol (10 mL) using a separatory funnel, the resulting butanol layerwas concentrated under reduced pressure and lyophilized to prepare asample. The yield was 5-15% and the prepared sample was kept at lowtemperature until use.

EXAMPLE 24 Preparation of Fractions of Seoritae (Glycin max MERR)Extract

Dried seoritae (1 kg) was immersed in 20% ethanol solution (10 L) at 50°C. After extracting 3 times for 5 hours under reflux and allowing tostand at room temperature for 12 hours, the extract was filtered andconcentrated under reduced pressure.

After adding 5 times the volume of ethyl acetate solution to theconcentrated filtrate and allowing to stand at room so that separationoccurs between an ethyl acetate and a water layer, only the ethylacetate layer was taken and lyophilized to prepare fractions. Theprepared powder was kept at low temperature until use.

COMPARATIVE EXAMPLE Preparation of Seoritae (Glycin max MERR) ExtractUsing Water

Dried seoritae (1 kg) was immersed in 20% water (10 L) at 100° C. Afterextracting 3 times for 5 hours under reflux and allowing to stand atroom temperature for 12 hours, the extract was filtered, concentratedunder reduced pressure and lyophilized to prepare a powder sample. Theyield was 7-20% and the prepared powder was kept at low temperatureuntil use.

TEST EXAMPLE 1 Inhibition of Human Platelet Aggregation Induced byCollagen of Seoritae (Glycin max MERR) Extract

Experiment was performed as follows in order to compare the activity ofthe extracts depending on ethanol concentration.

In order to isolate human platelet-rich plasma (hereinafter, PRP), bloodwas taken from the vein of a healthy man who did not take drugs over 2weeks using 3.2% sodium citrate as anticoagulant. The blood (150 g) wascentrifuged for 15 minutes. After separating the supernatant (PRP), theresidue was centrifuged again to separate platelet-poor plasma(hereinafter, PPP). The number of platelets in the separated PRP wascounted under an optical microscope, and the PRP was diluted with thePPP such that 3×10⁸ platelets were included per 1 mL.

The platelet aggregation activity was evaluated by measuring the changein absorbance using the lumi-aggregometer (Chrono-Log Co., USA). Afteradding 100 pg/mL of the seoritae extract to the PRP, followed byincubation in a thermomixer for 10 minutes, the incubated PRP (495 μL)was put in a silicone-coated cuvette for measurement of plateletaggregation and further incubated for 1 minute until the temperaturereached 37° C. Then, after adding collagen which induces plateletaggregation with a concentration of 5 μg/mL at which the maximumaggregation is achieved, the reaction was observed for 5 minutes. Theresult is given in Table 1. In Table 1, the inhibition of plateletaggregation (%) is a value relative to that of the control group treatedonly with collagen as 0%.

TABLE 1 Inhibition of platelet aggregation (%) Water extract 8.3 ± 2.510% ethanol extract 74.6 ± 6.4  20% ethanol extract 40.1 ± 12.2 50%ethanol extract 18.8 ± 10.9 70% ethanol extract 12.5 ± 8.4 

As seen from Table 1, the 10% and 20% ethanol extracts showed betterplatelet aggregation inhibition effect than other extracts. Especially,the 10% ethanol extract showed the best effect.

Also, an experiment was performed to identify the concentrationdependence of the platelet aggregation inhibition effect for the 20%ethanol extract. First, the same experiment was performed as describedabove after adding the extract at concentrations of 10, 25, 50 and 100μg/mL. The inhibition of platelet aggregation was evaluated relative tothat of the control group treated only with collagen as 0%. As seen fromFIG. 1, the 20% ethanol extract showed a concentration-dependentplatelet aggregation inhibition effect, demonstrating superior effectagainst blood clotting.

TEST EXAMPLE 2 Inhibition of Human Platelet Aggregation Induced byCollagen of Field Bean (Vida faba) Extract

Experiment was performed as follows in order to compare the activity ofthe extracts depending on ethanol concentration.

In order to isolate human PRP, blood was taken from the vein of ahealthy man who did not take drugs over 2 weeks using 3.2% sodiumcitrate as anticoagulant. The blood (150 g) was centrifuged for 15minutes. After separating the supernatant (PRP), the residue wascentrifuged again to separate PPP. The number of platelets in theseparated PRP was counted, and the PRP was diluted with the PPP suchthat 3×10⁸ platelets were included per 1 mL.

The platelet aggregation activity was evaluated by measuring the changein absorbance using the lumi-aggregometer (Chrono-Log Co., USA). Afteradding 200 mg/mL of the field bean extract to the PRP, which had beenincubated in a thermomixer at 37° C. for 2 minutes, followed byincubation for 7 minutes, the incubated PRP (500 μL) was put in asilicone-coated cuvette for measurement of platelet aggregation andfurther incubated for 3 minutes. Then, after adding collagen whichinduces platelet aggregation with a concentration of 1-3 μg/mL, i.e. thelowest concentration at which the maximum aggregation is achieved, thereaction was observed for 6 minutes. The result is given in Table 2. InTable 2, the inhibition of platelet aggregation (%) is a value relativeto that of the control group treated only with collagen as 0%.

TABLE 2 Inhibition of platelet aggregation (%) Water extract 69.0 10%ethanol extract 57.3 20% ethanol extract 76.3 30% ethanol extract 38.740% ethanol extract 34.7 50% ethanol extract 39.7 70% ethanol extract10.7

As seen from Table 2, the 10% and 20% ethanol extracts showed betterplatelet aggregation inhibition effect than other extracts. Especially,the 20% ethanol extract showed the best platelet aggregation inhibitioneffect.

TEST EXAMPLE 3 Human Platelet Aggregation Inhibition Effect of DifferentBeans

Under the 20% ethanol extraction condition showing the best activity,experiment was performed as follows in order to investigate the plateletaggregation inhibition of black bean. All the beans used in theexperiment were produced in Korea.

Experiment was performed for seoritae, seomoktae and Black soybean. Thebeans were extracted in the same manner as in Example 2. In order toinvestigate the platelet aggregation inhibition effect, experiment wasperformed using 100 μg/mL bean extract under the same condition as inTest Example 1. The result is given in Table 3.

TABLE 3 Beans Inhibition of platelet aggregation (%) Seoritae 45.31 ±9.67 Seomoktae  42.86 ± 11.02 Black soybean 41.78 ± 8.49

As seen from Table 3, the black beans seoritae, seomoktae and heuktaeshowed high platelet aggregation inhibition effect. Accordingly, it wasconfirmed that the low-concentration ethanol extracts of seoritae,seomoktae and heuktae have superior platelet aggregation inhibitioneffect.

TEST EXAMPLE 4 Human Platelet Aggregation Inhibition Effect of DifferentBeans

Under the 20% ethanol extraction condition showing the best activity,experiment was performed as follows in order to investigate the plateletaggregation inhibition of black bean. All the beans used in theexperiment were produced in Korea.

Experiment was performed for small black bean, soybean, seoritae,seomoktae, field bean, kidney bean, small red bean, blue bean, sproutingbean and yellow bean. The beans were extracted in the same manner asdescribed above. In order to investigate the platelet aggregationinhibition effect, experiment was performed using 200 μg/mL bean extractunder the same condition as in Test Example 2. The result is given inTable 4.

TABLE 4 Beans Inhibition of platelet aggregation (%) Small black bean31.0 Soybean 19.3 Seoritae 65.3 Field bean 76.3 Kidney bean 31.0 Smallred bean 32.0 Blue bean 16.5 Sprouting bean 37.5 Yellow bean 2.5

As seen from Table 4, seoritae, seomoktae and field bean showed superiorplatelet aggregation inhibition effect.

TEST EXAMPLE 5 Inhibition Specificity for Different Causes of PlateletAggregation

Under the 20% ethanol extraction condition, it was investigated whetherthe seoritae extract show specific inhibition effect for differentcauses of platelet aggregation.

It is known that platelet aggregation is induced by collagens which areexposed to the bloodstream when a blood vessel is damaged, ADPs secretedby the platelets, stress by the bloodstream (shear stress; hereinafter,SS), and thrombins. Accordingly, it was investigated whether the 20%low-concentration ethanol seoritae extract has inhibition specificityfor collagen, ADP, thrombin and shear stress (SS). The evaluation wasperformed in the same manner as in Test Example 1. ADP, thrombin orshear stress was applied instead of collagen. The result is given inTable 5.

TABLE 5 Collagen ADP Thrombin Shear stress Inhibition 61.38 ± 10.44 8.25± 3.10 19.61 ± 4.52 12.36 ± 3.68 of platelet aggregation (%)

As seen from Table 5, the 20% ethanol seoritae extract specificallyinhibited platelet aggregation induced by collagen, with little effectof inhibiting platelet aggregation induced by other stimulations, i.e.ADP, thrombin or shear stress.

TEST EXAMPLE 6 Active Substance Expression and Secretion InhibitionEffect After Platelet Aggregation

When platelets are coagulated by stimulation such as collagen, theyexpress specific proteins on their surface or secrete specificsubstances out of cells. Accordingly, it was investigated whether the20% low-concentration ethanol seoritae extract can reduce the expressionof P-selectin and secretion of serotonin into the bloodstream bysuppressing platelet aggregation.

After adding the sample to the same PRP as that of Test Example 1,followed by incubation for 10 minutes in an incubator and reaction for 6minutes after adding 10 μg/mL of collagen, the extract was added to thetest tube together with anti-CD42b-PE and anti-CD62P-FITC and allowed toreact for 20 minutes in the shade. Then, Tyrode's buffer (500 μL) wasadded to terminate the reaction.

Measurement was made using a fluorescence-activated cell sorter (FACS;BD Bioscience, USA). The degree of P-selectin expression was determinedby measuring the reduction of fluorescence due to the expression ofP-selectin. The result is given in Table 6. The inhibition of P-selectinexpression is given relative to the fluorescence of theextract-untreated group as 100.

TABLE 6 Treatment concentration (μg/mL) 10 25 50 100 Inhibition ofP-selectin 13.8 ± 2.3 19.8 ± 1.6 24.1. ± 2.2 36.9 ± 1.9 expression (%)

As seen from Table 6, the 20% ethanol seoritae extract decreased theexpression of P-selectin on the surface of platelets in aconcentration-dependent manner.

The secretion of serotonin was measured using the radioisotope method.After adding 0.5 μCi/mL of ¹⁴C-serotonin (Amersham Bioscience, CFA170)to the same PRP as that of Test Example 1, followed by treatment at 37°C. for 45 minutes and further treatment at 37° C. for 10 minutes afteradding the extract, 2 μg/mL of collagen was added and allowed to reactfor 6 minutes. After terminating the reaction by adding EDTA,centrifugation was carried out at 12000×g for 2 minutes. The quantity ofsecreted of [¹⁴C]-serotonin in the supernatant was measured using aliquid scintillation counter (Wallac 1409, Perkin Elmer, USA). Theinhibition of serotonin secretion was calculated relative to that of theextract-untreated group as 100. The result is given in Table 7.

TABLE 7 Treatment concentration (μg/mL) 10 25 50 100 Inhibition ofserotonin 9.8 ± 2.5 17.7 ± 3.2 38.0. ± 2.2 59.4 ± 1.3 secretion (%)

As seen from Table 7, the 20% ethanol seoritae extract decreased thesecretion of serotonin into the bloodstream in a concentration-dependentmanner.

TEST EXAMPLE 7 Inhibition of Blood Clotting in Vein in SD Rat

The blood clotting inhibition effect of the 20% ethanol seoritae extractin vivo was investigated as follows. Male Sprague Dawley (hereinafter,SD) rats weighing 220-250 g were used.

0, 50 or 100 μg/mL of the seoritae extract dissolved in saline (300 μL)was orally administered to the SD rat. 1 hour later, sodiumpentobarbital (50 mg/kg) was abdominally administered and the rat wasgenerally anesthetized. After cutting the abdomen open, the adiposetissue was removed so that the caudal vena cava could be seen well. Carewas taken to avoid damage to the nearby blood vessels when removing theadipose tissue. A filter paper soaked with 5% FeCl₃ solution was put onthe caudal vena cava for 5 minutes and then removed. 30 minutes later,the caudal vena cava containing blood clots was ligated at 12 mm lengthand then incised. After transferring the blood clots to saline, waterwas removed and the weight was measured. The result is given in FIG. 2.

Referring to FIG. 2, it can be seen that the oral administration of the100 mg/kg seoritae extract significantly inhibits blood clotting.

Also, the 50 or 100 mg/kg extract was orally administered for 14 days,and the result is shown in FIG. 3. Differently from the result 1 hourafter the administration, both the 50 and 100 mg/kg extractsignificantly inhibited blood clotting. Accordingly, it can be seen thatthe seoritae extract extracted using the low-concentration ethanol is asuperior anticoagulant.

TEST EXAMPLE 8 Human Platelet Aggregation Inhibition Effect of SeoritaeExtract

It was investigated which fraction exhibits superior activity under the20% ethanol extraction condition.

First, ethyl acetate, butanol and water fractions were prepared in thesame manner as in Example 25. The platelet aggregation inhibition effectof each fraction (100 μg/mL) was evaluated in the same manner as in TestExample 1. The result is given in Table 8.

TABLE 8 Inhibition of platelet aggregation (%) Ethyl acetate fraction95.2 ± 0.9 Butanol fraction 76.8 ± 6.9 Water fraction  4.7 ± 5.2

As seen from Table 8, the ethyl acetate fraction showed the bestplatelet aggregation inhibition effect, followed by butanol and waterfractions. Accordingly, it though that the platelet aggregationinhibition effect of the low-concentration ethanol extract is derivedfrom the active ingredients included in the ethyl acetate fraction.

In the ethyl acetate fraction that exhibited the best effect, theactivity was concentration-dependent, as shown in FIG. 4. That is tosay, the platelet aggregation inhibition effect increased in the orderof 10, 25, 50 and 100 μg/mL of the ethyl acetate fraction, as seen fromFIG. 4.

TEST EXAMPLE 9 Platelet Aggregation Inhibition Effect of ButanolFractions of Different Beans

Experiment was performed as follows in order to identify the plateletaggregation inhibition effect of the butanol fraction of differentbeans.

The platelet aggregation inhibition effect of the butanol fraction (200μg/mL) of soybean, seomoktae, seoritae, field bean, kidney bean, bluebean, sprouting bean and yellow bean was evaluated in the same manner asin Test Example 2. The result is given in Table 9.

TABLE 9 Inhibition of platelet aggregation (%) Soybean 94.0 Seomoktae90.5 Seoritae 72.7 Field bean 88.5 Kidney bean 77.5 Blue bean 91.5Sprouting bean 90.5 Yellow bean 88.5

As seen from Table 9, the butanol fractions of all of the beans showedsuperior platelet aggregation inhibition effect.

TEST EXAMPLE 10 Specificity of Platelet Aggregation Inhibition of EthylAcetate Fraction for Different Causes

Experiment was performed in the same manner as in Test Example 5 toinvestigate the specificity of platelet aggregation inhibition of ethylacetate fractions (50 and 100 μg/mL) for different causes. The result isgiven in Table 10.

TABLE 10 Shear Conc. Collagen ADP Thrombin stress Inhibition  50 μg/mL80.6 ± 0.8 25.1 ± 9.2 0.6 ± 3.3 16.0 ± 2.3 of platelet 100 μg/mL 94.0 ±1.9 53.7 ± 6.3 9.1 ± 7.7 36.9 ± 3.0 aggre- gation (%)

As seen from Table 10, the ethyl acetate fraction specifically inhibitedthe activity of collagen in a concentration-dependent manner.

TEST EXAMPLE 11 Inhibition Effect of Ethyl Acetate Fraction AgainstExpression and Secretion of Active Substances After Platelet Aggregation

Experiment was performed in order to investigate the ethyl acetatefraction of seoritae of reducing expression of P-selectin, secretion ofserotonin and generation of thromboxane after platelet aggregation.Experiment for P-selectin and serotonin was performed in the same manneras in Test Example 6. The inhibition of thromboxane generation wasevaluated by adding the ethyl acetate fraction to the PRP used in TestExample 1, incubating at 37° C. for 10 minutes, adding collagen (10μg/mL) and then further incubating for 6 minutes. Some of the sample wasadded to a test tube containing EDTA and indomethacin (final conc.=50μM) to terminate the reaction. After centrifuging at 12000×g for 2minutes, the supernatant was subjected to enzyme immunoassay forquantification of the produced thromboxane.

The inhibition of P-selectin expression by the ethyl acetate fraction isshown Table 11, the inhibition of serotonin secretion in Table 12, andthe inhibition of thromboxane generation in Table 13.

TABLE 11 Concentration (μg/mL) 10 25 50 100 Inhibition of P-selectin16.2 ± 2.3 39.0 ± 1.5 58.8. ± 0.3 73.7 ± 0.2 expression (%)

TABLE 12 Concentration (μg/mL) 10 25 50 100 Inhibition of serotonin 12.3± 2.3 40.3 ± 2.0 66.8 ± 3.1 89.6 ± 0.6 secretion (%)

TABLE 13 Concentration (μg/mL) 10 25 50 100 Inhibition of 2.6 ± 0.5 5.4± 3.7 11.4. ± 3.2 38.3 ± 3.6 thromboxane generation (%)

As seen from Tables 11, 12 and 13, the ethyl acetate fraction ofseoritae reduced the expression of P-selectin, inhibited the secretionof serotonin and inhibited the generation of thromboxane after plateletaggregation, in a concentration-dependent manner.

TEST EXAMPLE 12 Inhibition of Ethyl Acetate Fraction Against BloodClotting in Vein of SD Rat

Experiment was performed in the same manner as in Test Example 7 inorder to investigate whether the administration of the ethyl acetatefraction actually provides blood clotting inhibition effect in vivo. Theoral administration dose was 10, 25 and 50 mg/kg, and the amount ofproduced blood clots was determined 1 hour after the administration. Theresult is shown in FIG. 5.

As seen from FIG. 5, the oral administration of the ethyl acetatefraction inhibited blood clotting in a concentration-dependent manner.

TEST EXAMPLE 13 Inhibition Effect Against Aggregation of PlateletsDerived from SD Rat

Experiment was performed in the same manner as in Test Example 1 inorder to investigate whether the low-concentration ethanol extract andthe ethyl acetate fraction of seoritae is effective in inhibiting theaggregation of platelets derived from SD rat. The treatmentconcentration was 10, 25, 50 and 100 μg/mL, and the result is given inTable 14.

TABLE 14 Concentration (μg/mL) 10 25 50 100 Inhibition of Low-  3.7 ±1.2 17.1 ± 2.3 36.3 ± 5.7 46.5 ± 5.4 platelet concen- aggregationtration extract (%) Ethyl 17.1 ± 6.9 48.6 ± 3.8 66.1 ± 2.9 80.0 ± 4.4acetate fraction

As seen from Table 14, not only the ethanol low-concentration extractbut also the ethyl acetate fraction showed very superior effect ofinhibiting the aggregation of platelets derived from SD rat, in aconcentration-dependent manner.

TEST EXAMPLE 14 Observation of Vasoconstriction Inhibition Effect UsingBlood Vessel Ring

Male SD rats for experiment weighing 250-300 g were acquired from DaehanBiolink (Seoul, Korea) and kept under the condition of 22±2° C. and45-55% humidity, with 12/12-hr light/dark cycles (light from 7 a.m. to 7p.m.). Feed (Purina Korea, Seoul, Korea) and water were given freelyduring an accommodation period of 1 week.

After sacrificing the rat by bleeding, the chest was cut open and thethoracic aorta was taken out quickly and transferred to KR buffer[composition (mM): NaCl 115.5, KCl 4.6, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂2.5, NaHCO₃ 25.0, Ca²⁺ EDTA 0.026 glucose 11.1; pH 7.4] saturated with95% O₂/5% CO₂ mixture gas. A 3-4 mm long blood vessel ring was preparedby removing the blood and neighboring adipose and connective tissues inthe blood vessel. After applying tension gradually to the blood vesselring for initial 30 minutes until the equilibrium was reached, it wasconstricted with 10⁻⁶ M phenylephrine and then dilated with 10⁻⁶ Macetylcholine. The one with vasodilation of 80% or more was used.Maximum vasoconstriction was induced by replacing the buffer in a waterbath with KR buffer containing 90 mM KCl saturated with 95% O₂/5% CO₂mixture gas. After pretreating the blood vessel for 30 minutes with theseoritae extract at different concentrations, vasoconstriction wasinduced in the water bath with phenylephrine of gradually increasingconcentrations. The vasoconstriction induced by phenylephrine and 90 mMKCl is shown in Table 15 and Table 16.

TABLE 15 Phenylephrine Seoritae extract concentration (μM) Control(constriction %) (constriction %) 0.1 15.26 ± 7.04  0.39 ± 0.27 1 60.35± 4.83 16.30 ± 3.44 10 92.13 ± 8.46 40.02 ± 7.57 100  97.69 ± 10.54 53.68 ± 10.08

As seen from Table 15, the vasoconstriction increased with theconcentration of the phenylephrine. The treatment with the seoritaeextract at 100 μg/mL resulted in a concentration-dependentvasoconstriction inhibition effect.

TABLE 16 Seoritae extract (μg/mL) Control 25 50 Constriction (%) 87.55 ±16.01 70.45 ± 9.56 59.6 ± 6.69

As seen from Table 16, the treatment with the seoritae extract at 25 and50 μg/mL resulted in a concentration-dependent vasoconstrictioninhibition effect. Accordingly, it can be seen that the 20% ethanolseoritae extract provides very superior vasoconstriction inhibitioneffect.

TEST EXAMPLE 15 Lowering of Serum and Liver Lipid Levels in Animal Model

8-week-old female rats weighing 250-300 g were kept in polycarbonatecages, 8 heads per cage, maintained at 22±2° C. and 55±15% relativehumidity with 12/12-hr light/dark cycles. Normal diet orhigh-cholesterol diet was provided and water was given freely.

Fenofibric acid (200 mg/kg) used for treatment of hyperlipidemia wassuspended in 1% methyl cellulose (MC) and orally administered as apositive control. The seoritae extract (200 mg/kg) was orallyadministered once a day for 4 weeks. After fasting for 12 hours, bloodsample was taken from the retro-orbital plexus and centrifuged for 10minutes at 10000 rcf (relative centrifugal force). After thecentrifugation, total cholesterol, LDL-cholesterol, HDL-cholesterol andtriglyceride levels in the obtained serum were evaluated.

The analysis was performed using an automated hematology analyzer andthe Roche diagnostic kit. The result is shown in Table 17 (serum lipids)and Table 18 (liver lipids).

TABLE 17 (mg/ Seoritae dL) Normal Control Positive control extract Total114.25 ± 2.41  218.57 ± 20.44 112.42 ± 13.33 156.42 ± 10.21 cho- les-terol HDL- 84.85 ± 0.35 55.72 ± 3.56 87.89 ± 6.84 50.35 ± 6.88 cho- les-terol LDL-  30.5 ± 6.42 134.75 ± 9.41  75.34 ± 2.80  97.21 ± 10.44 cho-les- terol

As seen from Table 17, the control group rats fed with thehigh-cholesterol diet for 4 weeks showed about 2 times higher totalcholesterol level and about 4.4 times higher LDL-cholesterol level inserum as compared to the normal group. This shows that hyperlipidemiawas induced well by the high-cholesterol diet. The group treated withthe seoritae extract as well as the high-cholesterol diet showed about28% decreased total cholesterol level (156.42 mg/dL) and about 28%decreased LDL-cholesterol level as compared to the control group.

TABLE 18 Positive Seoritae (μg/mg) Normal Control control extract Total 5.81 ± 0.85 27.96 ± 2.46 15.43 ± 0.52 13.50 ± 7.62 cholesterolTriglyceride 199.91 ± 24.33 230.35 ± 10.53 185.39 ± 23.84 172.32 ± 11.16

Total cholesterol and triglyceride levels in the liver are shown inTable 18. It was confirmed that fatty liver was induced by thehigh-cholesterol diet in the control group. The seoritae extract-treatedgroup showed 52% decreased total cholesterol level and 25% decreasedtriglyceride level. Accordingly, it was confirmed that the seoritaeextract has superior effect of improving serum and liver lipid levels invivo.

TEST EXAMPLE 16 Analysis of Active Ingredient in Seoritae ExtractFractions

In order to analyze the active ingredient of the seoritae extract,assay-guided fractionation was carried out for the ethyl acetatefractions. The overall scheme of isolating and purifying the activeingredient of the ethyl acetate fraction is shown in FIG. 6.

Referring to FIG. 6, solid-phase extraction was carried out for theethyl acetate fraction A-2 of the 20% ethanol seoritae extract obtainedin Example 25. During the solid-phase extraction, 50-100% (v/v) methanol(MeOH) solution was used as the extraction solvent. The solid-phaseextracted extract was fractionated to A-3 through A-9. Plateletaggregation was evaluated for the extract, and the result is shown inFIG. 7. As seen from FIG. 7, the extracts A-3 and A-4 showed relativelylow platelet aggregation.

A mixture of the extracts A-3 and A-4 that showed relatively lowplatelet aggregation was subjected to column chromatography (Sephadex LH20 column chromatography) for separation of the components. Theseparated components were numbered from A-3-1 to A-3-10. Plateletaggregation was evaluated again for A-3-1 through A-3-10, and the resultis shown in FIG. 8. As seen from FIG. 8, the sample A-3-1 showed thelowest platelet aggregation among A-3-1 through A-3-10.

The sample A-3-1 that showed the lowest platelet aggregation wassubjected to high-performance liquid chromatography (preparative ODSHPLC) for separation of the active ingredients. The separated componentswere numbered from A-3-1-1through A-3-1-7. Platelet aggregation wasevaluated for the components, and the result is shown in FIG. 9. As seenfrom FIG. 9, A-3-1-3 showed the lowest platelet aggregation. Toconclude, A-3-1-3 showed the best platelet aggregation inhibitionactivity.

The platelet aggregation inhibition activity of A-3-1-3 was comparedwith that of standard adenosine. The result is shown in FIG. 10. As seenfrom FIG. 10, A-3-1-3 showed almost the same effect as that ofadenosine, suggesting that A-3-1-3 comprises adenosine. The chemicalstructure of adenosine is as follows:

TEST EXAMPLE 17 Measurement of Adenosine Content in 20% Ethanol SeoritaeExtract

The activity of the 20% ethanol seoritae extract was compared with thatof standard adenosine, and the result is shown in FIG. 11 and FIG. 12.Referring to FIG. 11 and FIG. 12, the content of adenosine in the 20%ethanol seoritae extract is calculated as 0.35-0.5% from the comparisonwith the standard adenosine. That is to say, the seoritae extractextracted using the low-concentration, lower alcohol has a very highadenosine content. In addition, the blood clotting inhibition of theseoritae extract is better than that of adenosine alone. Accordingly, amore stable inhibition of blood clotting can be expected from theseoritae extract as compared to when adenosine is used alone.

TEST EXAMPLE 18 Measurement of Adenosine Content in 20% Ethanol BeanExtracts

Adenosine content in the 20% ethanol extracts of various beans includingseoritae was measured. The result is given in Table 19.

TABLE 19 Beans Adenosine content (%) Seoritae 0.36 Seomoktae 0.20 Bluebean 0.19 Yellow bean 0.16 Kidney bean 0.29 Sprouting bean 0.15 Fieldbean 0.34

As seen from Table 19, the adenosine content was high with 0.15% orabove in various beans. In particular the seoritae and field beanextracts showed relatively high adenosine content.

TEST EXAMPLE 19 Blood Clotting Inhibition Activity of Seoritae (Glycinmax MERR) Extract in Red Blood Cells

It was investigated whether the bean extract affects the exposure of redblood cells to phosphatidylserine (hereinafter, PS) and the generationof microvesicles (hereinafter, MV) by lysophosphatidic acid(hereinafter, LPA) and phosphatidic acid (hereinafter, PA) which areproduced during inflammation.

In general, it is known that blood clotting is facilitated when redblood cells are exposed to PS or MV is generated. After treating withthe seoritae extract in inflammatory state, inhibition of PS exposureand MV generation induced by LPA and PA were measured. Specifically, 1hour after adding the seoritae extract (100 μg/mL), 50 μM LPA or 25 μMPA was added for 15 minutes to induce PS exposure and MV generation.Then, the degree of PS exposure and MV generation was compared betweenthe case where the seoritae extract was treated (+) and untreated (−).The degree of PS exposure is shown in FIG. 13, and the degree of MVgeneration is shown in FIG. 14.

As seen from FIGS. 13 and 14, the seoritae extract inhibited the PSexposure and MV generation induced by LPA and PA.

Further, the degree of thrombin generation was measured byprothrombinase assay from the change in procoagulant activity mediatedby PS exposure. The result is shown in FIG. 15. In the figure, (+) isthe case where the seoritae extract was treated, and (−) is the casewhere it was untreated. As seen from FIG. 15, the treatment with theseoritae extract inhibited the thrombin generation by LPA and PA.

TEST EXAMPLE 20 Inhibition of Blood Clotting by Seoritae Extract

Coagulation is a procedure whereby blood clots are formed as coagulationfactors which are zymogens are sequentially activated to turn fibrinogento fibrin. An intrinsic pathway also called the contact system and anextrinsic pathway initiated by the activation of tissue factors lead tothe formation of blood clots.

The effect of the 20% ethanol seoritae extract on the extrinsic pathwayor the intrinsic pathway of the coagulation cascade was evaluated by theprothrombin time (PT) and the activated partial thromboplastin time(hereinafter, aPTT).

PT was measured as follows. Blood was taken from the rat and centrifugedusing 3.8% sodium citrate as anticoagulant. After adding RecombiPlasTinto the supernatant, the time to fibrin formation was measured using afibrinometer. A longer time means better inhibition of blood clotting.

aPTT was measured as follows. Blood was taken from the rat andcentrifuged using 3.8% sodium citrate as anticoagulant. After adding 20mM calcium chloride (CaCl₂) solution to the supernatant, 100 μL of theserum was taken and the time to fibrin formation was measured using afibrinometer.

The PT measurement result is shown in FIG. 16, and the aPTT measurementresult is shown in FIG. 17.

As seen from FIG. 16 and FIG. 17, the 20% ethanol seoritae extract hadno effect on PT or aPTT. Accordingly, since the seoritae extract doesnot affect the normal coagulation of blood, it will not result in theside effects of the known anticoagulants (e.g., aspirin) such asinterruption of hemostasis or excessive bleeding.

TEST EXAMPLE 21 Effect of Seoritae (Glycin max MERR) Extract on BleedingTime

Bleeding time was compared for the cases where 20% ethanol seoritaeextract, aspirin or clopidogrel was treated. The result is shown in FIG.18.

As seen from FIG. 18, aspirin or clopidogrel resulted in significantincrease of bleeding time, whereas the seoritae extract resulted inlittle change in bleeding time. The bleeding time was almost unchangedeven when the treatment amount was increased 5-fold from 100 mg/kg to500 mg/kg. Accordingly, it can be seen that side effects such asincreased bleeding time do not occur even when the administration doseor treatment amount is increased.

Hereinafter, the formulation examples of compositions comprising thebean extract according to the present disclosure will be described indetail. The following examples are for illustrative purposes only andnot intended to limit the scope of the present disclosure.

FORMULATION EXAMPLE 1 Soft Capsule

Seoritae extract (100 mg) was mixed with soybean extract (50 mg),soybean oil (180 mg), red ginseng extract (50 mg), palm oil (2 mg),hydrogenated palm oil (8 mg), beeswax (4 mg) and lecithin (6 mg), andfilled in a soft capsule according to a commonly employed method.

FORMULATION EXAMPLE 2 Tablet

Field bean extract (100 mg) was mixed with soybean extract (50 mg),glucose (100 mg), red ginseng extract (50 mg), starch (96 mg) andmagnesium stearate (4 mg). After forming granules by adding 30% ethanol(40 mg), followed by drying at 60° C., a tablet was prepared using atablet making machine.

FORMULATION EXAMPLE 3 Granule

Seoritae extract (100 mg) was mixed with soybean extract (50 mg),glucose (100 mg), red ginseng extract (50 mg) and starch (600 mg). Afterforming granules by adding 30% ethanol (100 mg), followed by drying at60° C., the granules were filled in a pouch. The final weight was 1 g.

FORMULATION EXAMPLE 4 Drink

Seoritae extract (100 mg) was mixed with soybean extract (50 mg),glucose (10 g), red ginseng extract (50 mg), citric acid (2 g) andpurified water (187.8 g), and filled in a bottle. The final volume was200 mL.

FORMULATION EXAMPLE 5 Health Food

Seoritae extract 1000 mg Vitamins Vitamin A acetate 70 μg Vitamin E 1.0mg Vitamin B₁ 0.13 mg Vitamin B₂ 0.15 mg Vitamin B₆ 0.5 mg Vitamin B₁₂0.2 μg Vitamin C 10 mg Biotin 10 μg Nicotinamide 1.7 mg Folic acid 50 μgCalcium pantothenate 0.5 mg Minerals Ferrous sulfate 1.75 mg Zinc oxide0.82 mg Magnesium carbonate 25.3 mg Potassium phosphate monobasic 15 mgCalcium phosphate dibasic 55 mg Potassium citrate 90 mg Calciumcarbonate 100 mg Magnesium chloride 24.8 mg

The exemplary contents of the vitamins and minerals in the health foodmay be changed as desired. The above ingredients were mixed and preparedinto granules according to the commonly employed health food preparationmethod for use in the preparation of the health food composition.

FORMULATION EXAMPLE 6 Health Drink

Field bean extract 1000 mg Citric acid 1000 mg Oligosaccharide 100 gConcentrated plum extract 2 g Taurine 1 g Purified water to make 900 mL

The above ingredients were mixed and heated at 85° C. for about 1 hourwith stirring according to the commonly employed health drinkpreparation method. The resulting solution was put in a sterilized 2-Lcontainer, sealed and sterilized, and then kept in a refrigerator foruse in the preparation of the health drink composition.

The composition of the health drink may be changed considering regionalor ethnic preferences, such as particular customers, country, purpose ofuse, or the like.

The composition according to the present disclosure may be widelyapplicable in the field of medicine, food and others.

1. A method for improving blood circulation of a subject, comprisingadministering to the subject an effective amount of a bean extractextracted with a C₁-C₅ alcohol with a concentration of 1-70% (v/v) or afraction thereof, wherein the bean extract or the fraction thereofimproves blood circulation in the subject.
 2. The method according toclaim 1, wherein the C₁-C₅ alcohol is at least one selected from a groupconsisting of methanol, ethanol, isopropyl alcohol, n-propyl alcohol,n-butanol and isobutanol.
 3. The method according to claim 2, whereinthe C₁-C₅ alcohol is ethanol.
 4. The method according to claim 1,wherein the bean extract or the fraction thereof comprises adenosine. 5.The method according to claim 4, wherein the adenosine is included in anamount of 0.01-1.0 wt % based on the weight of the bean extract or thefraction thereof.
 6. The method according to claim 1, wherein the beanis at least one selected from a group consisting of Seoritae, Seomoktae,Black soybean, blue bean, yellow bean, field bean, kidney bean, pintobean, small red bean, small black bean, sprouting bean and soybean. 7.The method according to claim 1, wherein the bean is Seoritae or fieldbean.
 8. The method according to claim 1, wherein the fraction is anethyl acetate or butanol fraction of a C₁-C₅ alcohol extract.
 9. Themethod according to claim 1, wherein the bean extract or the fractionthereof improves blood circulation through suppression of bloodclotting, suppression of vasoconstriction or suppression of cholesterolgeneration.
 10. The method according to claim 1, wherein the beanextract or the fraction thereof improves vascular health.
 11. (canceled)12. The method according to claim 1, wherein the method is forpreventing, alleviating or treating obesity, diabetes, stroke, cerebralhemorrhage, arteriosclerosis, angina, myocardial infarction,hypertension, anemia, migraine or hyperlipidemia.
 13. A health foodcomposition comprising a bean extract extracted with a C₁-C₅ alcoholwith a concentration of 1-70% (v/v) or a fraction thereof. 14.(canceled)
 15. The health food composition according to claim 13,wherein the bean is at least one selected from a group consisting ofSeoritae, Seomoktae, Black soybean, blue bean, yellow bean, field bean,kidney bean, pinto bean, small red bean, small black bean, sproutingbean and soybean.
 16. The health food composition according to claim 15,wherein the bean is Seoritae or field bean.
 17. The health foodcomposition according to claim 13, wherein the fraction is an ethylacetate or butanol fraction of a C₁-C₅ alcohol extract.
 18. Apharmaceutical composition comprising a bean extract extracted with aC₁-C₅ alcohol with a concentration of 1-70% (v/v) or a fraction thereof.19. The pharmaceutical composition according to claim 18, wherein thebean is at least one selected from a group consisting of Seoritae,Seomoktae, Black soybean, blue bean, yellow bean, field bean, kidneybean, pinto bean, small red bean, small black bean, sprouting bean andsoybean.
 20. The pharmaceutical composition according to claim 19,wherein the bean is Seoritae or field bean.
 21. The pharmaceuticalcomposition according to claim 18, wherein the fraction is an ethylacetate or butanol fraction of a C₁-C₅ alcohol extract.